Mechanism of N 2 Reduction Catalyzed by Fe-Nitrogenase Involves Reductive Elimination of H 2

Of the three forms of nitrogenase (Mo-nitrogenase, V-nitrogenase, and Fe-nitrogenase), Fe-nitrogenase has the poorest ratio of N2 reduction relative to H2 evolution. Recent work on the Mo-nitrogenase has revealed that reductive elimination of two bridging Fe−H−Fe hydrides on the active site FeMo-cofactor to yield H2 is a key feature in the N2 reduction mechanism. The N2 reduction mechanism for the Fe-nitrogenase active site FeFe-cofactor was unknown. Here, we have purified both component proteins of the Fe-nitrogenase system, the electrondelivery Fe protein (AnfH) plus the catalytic FeFe protein (AnfDGK), and established its mechanism of N2 reduction. Inductively coupled plasma optical emission spectroscopy and mass spectrometry show that the FeFe protein component does not contain significant amounts of Mo or V, thus ruling out a requirement of these metals for N2 reduction. The fully functioning Fe-nitrogenase system was found to have specific activities for N2 reduction (1 atm) of 181 ± 5 nmol NH3 min −1 mg−1 FeFe protein, for proton reduction (in the absence of N2) of 1085 ± 41 nmol H2 min −1 mg−1 FeFe protein, and for acetylene reduction (0.3 atm) of 306 ± 3 nmol C2H4 min−1 mg−1 FeFe protein. Under turnover conditions, N2 reduction is inhibited by H2 and the enzyme catalyzes the formation of HD when presented with N2 and D2. These observations are explained by the accumulation of four reducing equivalents as two metal-bound hydrides and two protons at the FeFe-cofactor, with activation for N2 reduction occurring by reductive elimination of H2. N is the microbial enzyme responsible for biological dinitrogen (N2) fixation to ammonia (NH3) and represents the largest contributor of fixed nitrogen (N) to the global biogeochemical nitrogen cycle. There are only three known forms of nitrogenase designated as the molybdenum (Mo)-dependent, the vanadium (V)-dependent, and the iron (Fe)-dependent enzymes, each encoded by unique gene clusters, designated nif, vnf, and anf, respectively. Each nitrogenase contains a complex metal cluster called the FeMo-cofactor, FeV-cofactor, and FeFe-cofactor, as the active site for substrate reduction (Figure 1B). These multimetallic cofactors all contain Fe and S, with available evidence indicating that all three are similar, with substitution of one metal (Mo, V, or Fe). The X-ray structure of the Mo-nitrogenase has been well established, and a recent X-ray structure of the V-dependent nitrogenase confirms that the basic architectures of FeMo-cofactor and FeV-cofactor are similar, with V substituting for Mo. Additionally, the structure of FeV-cofactor shows a molecule replacing one of the bridging sulfide atoms found in FeMo-cofactor. While the identity of the molecule replacing S is unknown, it has been proposed to be carbonate. No structural information is available for the FeFecofactor in Fe-nitrogenase. Mo-nitrogenase is the best studied of the three nitrogenase forms. It is a two-component system comprising a catalytic MoFe protein (NifDK) and electron-delivery Fe protein (NifH) (Figure 1A). The Fe protein is a homodimer that contains a single Fe4S4 cluster and two MgATP-binding sites. 5 The MoFe protein is a α2β2 heterotetramer that forms two catalytic halves, with each half containing a Fe8S7 cluster (Pcluster) and a MoFe7S9C-homocitrate active site cofactor (FeMo-co). During catalysis, the two component proteins transiently associate, during which the Fe protein donates one electron to the MoFe protein, coupled to the hydrolysis of two MgATP. Electrons are accumulated on FeMo-cofactor, with the P-cluster proposed to act as a “deficit-spending” electron shuttle between the Fe protein and FeMo-cofactor. Studies of the MoFe protein trapped during catalysis have shown how electrons and protons accumulate on FeMo-cofactor and how the enzyme is activated for N2 binding and reduction (Figure 1C). In short, four electrons and protons must be accumulated on FeMo-cofactor to create the E4(4H) state before N2 can be reduced. This E4(4H) state contains two Fe− H−Fe bridging hydrides. The two hydrides combine to make H2 in a reductive elimination (re) reaction that is coupled to N2 binding and reduction by two electrons/protons to the first bound intermediate, a diazenido-metal complex (E4(2N2H)). The re step is reversible, with the oxidative addition (oa) of H2 by E4(2N2H) leading to N2 release. 13,16−19 The reversibility of this re/oa step explains early findings that two HD are formed for every D2 consumed when nitrogenase is turned over in the presence of D2 and N2. 20,21 This observation can be understood from the mechanistic model as resulting from oa of D2 by the E4(2N2H) state, which leads to formation of two metal-bound D− with loss of N2. These nonexchangeable deuterides each react with H derived from solvent, yielding two HD. Overall, the two signature features of the re/oa mechanism for N2 activation by nitrogenase are the observations that H2 can inhibit N2 reduction and that turnover in the presence of N2 and D2 results in the formation of HD. Studies on V-nitrogenase have indicated that while it does reduce N2, it does so at a lower rate compared to Monitrogenase, and little is known about its catalytic mechanism. Even fewer studies have been conducted on Fenitrogenase, and less is known about its mechanism for N2 reduction. Like Mo-nitrogenase, Fe-nitrogenase comprises an electron-delivery Fe protein (AnfH) and catalytic “FeFe protein” (AnfDGK) (Figure 1A). Compared to the MoFe protein, the FeFe protein incorporates an extra gamma subunit per catalytic half, forming an α2β2γ2 heterohexamer (Figure 1A). Amino acid residues in the Fe protein of Monitrogenase that coordinate the Fe4S4 cluster and nucleotide binding sites, as well as those in MoFe protein that coordinate the P-cluster and FeMo-cofactor, are conserved in the Fenitrogenase proteins. Spectroscopic studies predict that Fe-nitrogenase contains a metallocluster called FeFe-co that is structurally homologous to FeMo-co of Mo-nitrogenase. While structurally and functionally similar, some aspects of catalysis by the three nitrogenases are not identical. In the case of Mo-nitrogenase, the re/oa equilibrium incorporates a mechanistically required limiting stoichiometry (eq 1) for N2 reduction